Association analysis between CD14 gene polymorphisms and peri‐implantitis susceptibility in a Chinese population

Abstract Objective The goal of the study was to examine the genetic correlation of cluster of differentiation 14 (CD14) gene polymorphisms with peri‐implantitis (PI) predisposition in a Chinese Han population. Methods In the case‐control study, blood samples were collected from PI patients and healthy individuals (n = 120/group), who were admitted to the Affiliated Hospital of Yangzhou University from 2021 to 2023. One‐way analysis of variance (ANOVA) was applied to compare differences of continuous variables among different groups. Genotype and allele distributions of CD14 gene rs2569190 and rs2915863 polymorphisms were analyzed between groups via χ 2 test. Results A high percentage of rs2569190 GG genotype or G allele carriers were identified in PI group compared with control group (p < .01). Rs2569190 GG genotype carriers had high risk to develop PI (odds ratio: 2.545, 95% confidence interval: 1.257–5.156, p = .009). The rs2569190 AA genotype carriers had the lowest values of gingival index, plaque index, calculus index, peri‐implant pocket depth, and clinical attachment level, which were the highest in cases with GG genotype. Conclusion Rs2569190 polymorphism of CD14 gene was significantly associated with PI predisposition in the Chinese Han population, and the GG genotype and G allele were risk factors for the development of PI.

develop PI. 2 But no effective therapeutic strategies have been explored against PI.PI is one of the most common complications of implant therpy, eventually leading to the loss of the functioning osseointegrated implants. 3hus, PI has been considered to be the leading cause of late implant failure along with occlusal overload. 4s a multifactorial disease, it can be regulated by pathogenic microorganisms and host inflammatory immune response.Various complex interactions including host factors, genetic and environmental factors are involved in the pathogenesis of PI. 5 Cluster of differentiation 14 (CD14) acts as a multifunctional high-affinity receptor for the binding of endotoxins, lipopolysaccharide (LPS).It is involved in primary immune and inflammatory responses. 6,7Additionally, LPS has been reported to be associated with the deterioration of gram-positive bacteria induce. 8CD14 can proportionally regulate the subsequent production of pro-inflammatory cytokines, eventually resulting in LPS-stimulated bone resorption. 7,9The CD14 expression has been determined to be involved in various pathological states, including periodontal diseases. 10Vijaya et al. 11 have also reported a significant association between elevated levels of CD14 and human periodontitis.The human CD14 gene is located on chromosome 5q31, and numerous single nucleotide polymorphisms (SNPs) have been detected in CD14 gene, which can mediate the protein's function.Recently, the relationship between CD14 gene polymorphisms and PI predisposition has been presented by Rakic et al. 12 The -1619A/G (rs2915863) and -159C/T (rs2569190) are two common SNP in the promoter region of the CD14 gene, and they are associated with altered levels of CD14. 13Previously, the two SNPs has been reported to be associated with the susceptibility of sepsis. 14Moreover, rs2569190 has been determined to be related to PI predisposition in Southeastern Europe Caucasians. 12However, their association with PI has not been evaluated in the Chinese Han population.
Therefore, the goal of this study was to examine the association of CD14 gene rs2569190 and rs2915863 polymorphisms with genetic susceptibility to PI in a Chinese Han population, which will be beneficial for the exploration of the pathogenesis of PI.

| Subjects
A total of 240 individuals were enrolled in this casecontrol study, including 120 patients with PI and 120 individuals with healthy implants.Cases in the PI group met the following inclusion criteria: (1) had at least one implant with the loading period longer than 12 months; (2) suppuration occurred on probing accompanied by bleeding and the probing depths greater than 5 mm; (3) one or more area presents crestal bone loss around the implant based on the Radiographic results, and at least two edges were exposed; (4) no occlusal trauma in the implant.The inclusion criteria of control group: (1) no history of periodontitis and clinical symptoms of periodontitis; (2) periodontal pocket depth is less than 3 mm; (3) no radiological findings of bone resorption.Exclusion criteria: (1) patients took immunological agents and antibiotics in the past 3 months; (2) patients undergoing orthodontic treatment; (3) patients with routine health problems such as hepatitis, diabetes and chemical therapy; (4) women during pregnancy and lactation.The patient's medical history was asked in detail, and their age, gender, body mass index, smoking history, drinking history, diet, and lifestyle habits and other basic information were recorded.
This study design was approved and consented by the Ethics Committee of Affiliated Hospital of Yangzhou University.Each participant signed and submitted a written informed consent.All individuals involved in this study were Chinese Han population and had no blood relationship with each other.

| Observation index
Before treatment, Williams periodontal probe was used for the oral examination, and gingival index, plaque index, calculus index, peri-implant pocket depth (PPD) and clinical attachment level (CAL) were recorded.

| Genotype identification
Approximately 10 mL of venous blood was drawn from each subject into a vacuum tube containing anticoagulant EDTA.DNA extraction kit (Tiangen Biochemical Technology Co., Ltd.) was applied for extraction of genomic DNA.Then the concentration and purity of the DNA samples were detected, and stored at −80°C for testing.
The target fragments were amplified by polymerase chain reaction, and the total volume of the amplification reaction was 40 μL, including 20 μL premix, 10 μL DNA template, 1 μL upstream primer, 1 μL downstream primers, and 8 μL distilled water.The reaction conditions were denatured at 98°C for 10 s, annealed at 63°C for 5 s, and extended at 72°C for 10 s, a total of 40 cycles were accomplished.After the reaction, the purified products were collected and sequenced by ABI 3500DX sequencer (ABI Corporation).

| Statistical analysis
SPSS 21.0 software was used for statistical analysis.For continuous variables, the data distributions were first evaluated for normality by the Kolmogorov-Smirnov test, and all variables did not conform to a normal distribution.So the data were expressed as median with interquartile range.The categorical variables were expressed as number and percent (%).The goodness-fitting χ 2 test was used to analyze the Hardy-Weinberg equilibrium.One-way analysis of variance (ANOVA) or χ 2 test was used to compare differences in general population data, periodontal clinical indicators and genotype distribution.Bilateral p < .05 was considered statistically significant.

| General characteristics of the study population
This case-control study was performed based on 240 patients receiving dental implants, among them 120 cases underwent PI while the remaining 120 individuals did not (Table 1).The control group consisted of 84 males (51.85%) and 78 females (48.15%) with the mean age and SD equal to 42.44 ± 5.92.Among the PI group, 89 (54.94%) cases were males and 73 (45.06%) individuals were females.The differences of both age and gender between the two groups were found nonsignificant (p > .05).The history of alcohol consumption, smoking and periodontitis also differed with no significant difference in the two groups (p > .05).The mouth washing habits, including frequencies of brushing, dental floss, and mouth washing had no prominent difference between the HC and PI groups (p > .05).Significant differences were found in respect to the periodontal status, it was shown that PI cases had high values of gingival index, plaque index, calculus index, PPD, and CAL (p < .05).But the platform type and position, perimplant phenotype exhibited no highlighted discrepancy between the HC and PI groups (p > .05).

| DISCUSSION
PI is an infectious disease affecting the soft and hard osseous tissue surrounding the implants, which is the main cause of the failure of the implant denture. 15ith the increasing demand for dental implants, the risk of dental implant failures appear to increase recently. 16Many factors can influence the occurrence of PI, including occlusal factors, general condition, smoking and oral health habit, and so on. 17When eliminated the possible risk factors, it is noted that most of PI occurs in a specific population, which is related to the host response to the bacterial challenge and susceptibility. 18Thus, host factor is essential in the occurrence of PI.Furthermore, patients had the history of one implant failure are much easier to failure of additional implants.And the susceptibility might be associated with genetic factors. 19D14 is a major endotoxin receptor which is implicated in the phagocytosis of bacteria and LPSmediated bone resorption. 20,21The CD14 expression can be activated by the stimulation of NF-κB and consequential upregulation of transcription of genes coding various pro-inflammatory cytokines. 22CD14 is a glyco-phosphatidylinositol-linked protein with the function of transferring LPS to toll-like receptors.Additionally, CD14 can affect subsequent production of pro-inflammatory cytokines, further resulting in LPSstimulated bone resorption. 23The human CD14 gene is a single-copy gene but encodes two distinctive protein forms.The membrane-bound CD14 receptor protein is mainly expressed on the surface of monocytes, macrophages, neutrophils, and gingival fibroblasts, it can participate in the process of cellular response to bacterial LPS.The other form, known as soluble form, is detected in monocyte or liver but lacks the anchor. 24CD14 has been considered as a causative gene for periodontitis, and Note: The data are represented as mean ± SD.
is implicated in periodontal tissue remodeling and degradation. 25n the current study, the CD14 gene two common polymorphisms namely, rs2569190 and rs2915863, were analyzed in the Chinese Han population.The results determined that the control group had a lower frequency of rs2569190 GG genotype than the PI group did.And the distribution differences reached a significant level.However, the AG genotype frequency of rs2569190 was almost equal between the two groups.All data implied the significant association of CD14 gene rs2569190 polymorphism with PI susceptibility in the Chinese Han population.And compared with AA genotype, the GG genotype carriers were much easier to suffer from PI. Rs2569190 polymorphism, also referred as CD14-159C/T, is a functional SNP located in the 5′ UTR of the promoter region of CD14 gene.It exerts considerable impact on the CD14 receptor, and then further influences the functionality of monocytes. 26As reported in a meta-analysis, CD14 -159C/T has been suggested to be involved in the development of periodontitis, which was in accordance with our present results. 27Furthermore, Rakic et al. 12 have reported a significant association between CD14 -159C/T polymorphisms and PI susceptibility, and rs2569190 may be a potential biomarker for PI, all evidence supported our present conclusion.In a study of sepsis, both C reactive protein and procalcitonin levels were higher among the AA homozygotes compared with the G-allele carriers, indicating that a significantly stronger pro-inflammatory response among AA patients compared with G-allele carriers. 28But according to present findings, the GG genotype carriers were much easier to suffer from PI. Considering the low proinflammatory response and high PI predisposition of GG genotype carriers, this may be attributed to immune suppression in individuals, which may be a risk factor for implant-related infection. 29However, the assumption needs to be confirmed.Rs2915863, also known as CD14 G-1619A, is another common SNP located on CD14 promoter region at position -1619. 30Rs2915863 polymorphism was also analyzed in this paper, but no significant association was detected with PI susceptibility in the Chinese Han population.

| CONCLUSION
In conclusion, our results showed that the rs2569190 polymorphisms of CD14 gene had a significant association with PI susceptibility in the Chinese Han population, and the GG genotype and G allele were risk factors for the occurrence of PI.Other studies were also needed to confirm our results due to the following reasons.First, a limited number of the study sample was recruited in this study, more eligible participants are feasible to reduce the risk of bias.Second, PI is a multifactorial disease, which is not affected by only one gene.Thus, other genes and polymorphisms should be considered.Thus other replication studies in various populations should also be performed.Additionally, an in-depth mechanism analysis is lacking, which should be considered in future studies.
General characteristics of the study population.
T A B L E 1 ***High statistical difference with the p-value less than .001.
Frequency distribution of CD14 gene rs2569190 and rs2915863 genotype and allele in HC and PI groups According to the Hardy-Weinberg exact test results, the included two study groups were all in equilibrium, with a p-value of more than .05(Table2).Based on the χ 2 Frequency distribution comparision of CD14 gene rs2569190 and rs2915863 genotype and allele in HC and PI groups based on χ 2 test.
T A B L E 3 Periodontal status in patients with CD14 gene rs2569190 different genotypes.Periodontal status in patients with different genotypes of CD14 gene rs2915863.